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Blood, Vol. 92 No. 11 (December 1), 1998:
pp. 4013-4022
RAPID COMMUNICATION
Highly Efficient Transduction of the Green Fluorescent Protein Gene in
Human Umbilical Cord Blood Stem Cells Capable of Cobblestone Formation
in Long-Term Cultures and Multilineage Engraftment of Immunodeficient
Mice
Paula B. van Hennik,
Monique M.A. Verstegen,
Marti F.A. Bierhuizen,
Ana Limón,
Albertus W. Wognum,
José A. Cancelas,
Jordi Barquinero,
Rob E. Ploemacher, and
Gerard Wagemaker
From the Institute of Hematology, Erasmus University Rotterdam, The
Netherlands; and the Department of Cryobiology and Cell Therapy,
Institut de Recerca Oncologica, Barcelona, Spain.
Purified CD34+ and
CD34+CD38 human umbilical cord blood (UCB)
cells were transduced with the recombinant variant of Moloney murine
leukemia virus (MoMLV) MFG-EGFP or with SF-EGFP, in which EGFP
expression is driven by a hybrid promoter of the spleen focus-forming virus (SFFV) and the murine embryonic stem cell virus (MESV). Infectious MFG-EGFP virus was produced by an amphotropic virus producer
cell line (GP+envAm12). SF-EGFP was produced in the PG13 cell
line pseudotyped for the gibbon ape leukemia virus (GaLV) envelope
proteins. Using a 2-day growth factor prestimulation, followed by a
2-day, fibronectin fragment CH-296-supported transduction, CD34+ and CD34+CD38 UCB
subsets were efficiently transduced using either vector. The use of the
SF-EGFP/PG13 retroviral packaging cell combination consistently
resulted in twofold higher levels of EGFP-expressing cells than the
MFG-EGFP/Am12 combination. Transplantation of 105 input
equivalent transduced CD34+ or 5 × 103
input equivalent CD34+CD38 UCB cells in
nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice resulted in median engraftment percentages
of 8% and 5%, respectively, which showed that the in vivo
repopulating ability of the cells had been retained. In addition, mice
engrafted after transplantation of transduced CD34+ cells
using the MFG-EGFP/Am12 or the SF-EGFP/PG13 combination expressed EGFP
with median values of 2% and 23% of human CD45+ cells,
respectively, which showed that the NOD/SCID repopulating cells were
successfully transduced. EGFP+ cells were found in all
human hematopoietic lineages produced in NOD/SCID mice including human
progenitors with in vitro clonogenic ability. EGFP-expressing cells
were also detected in the human cobblestone area-forming cell (CAFC)
assay at 2 to 6 weeks of culture on the murine stromal cell line
FBMD-1. During the transduction procedure the absolute numbers of CAFC
week 6 increased 5- to 10-fold. The transduction efficiency of this
progenitor cell subset was similar to the fraction of
EGFP+ human cells in the bone marrow of the NOD/SCID mice
transplanted with MFG-EGFP/Am12 or SF-EGFP/PG13 transduced
CD34+ cells, ie, 6% and 27%, respectively. The study
thus shows that purified CD34+ and highly purified
CD34+CD38 UCB cells can be transduced
efficiently with preservation of repopulating ability. The SF-EGFP/PG13
vector/packaging cell combination was much more effective in
transducing repopulating cells than the MFG-EGFP/Am12 combination.
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